Glucocerebroside treatment of disease

ABSTRACT

The present invention provides a method for the treatment of immune mediated or immune related diseases or disorders, infectious diseases, metabolic disorders and cancer in mammalian subjects. This method comprises the administration of a naturally occurring, mammalian intermediary metabolite or T cell receptor ligand, preferably a glucosylceramide, to a mammalian subject. In a preferred embodiment, such mammalian subjects are human beings.

REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/801,385 filed Feb. 26, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/174,532 filed Oct. 30, 2018, which is acontinuation of U.S. patent application Ser. No. 15/619,993 filed Jun.12, 2017, which is a continuation of U.S. patent application Ser. No.10/675,980 filed Sep. 30, 2003 (now U.S. Pat. No. 9,717,754), which is acontinuation-in-part of U.S. patent application Ser. No. 10/375,906filed on Feb. 27, 2003, each of which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to the use of a naturally occurring, mammalianintermediary metabolite or T cell receptor ligand, preferablyGlucocerebroside, for the treatment of immune mediated or immune relateddiseases or disorders, infectious diseases, metabolic disorders andcancer in mammalian subjects.

All patents, patent applications, patent publications, scientificarticles, and the like, are hereby incorporated by reference in theirentirety in order to describe more fully the state of the art to whichthe present invention pertains.

BACKGROUND OF THE INVENTION

Various methods have been described for the treatment of immune-relatedor immune mediated disorders or diseases, infectious diseases, metabolicdisorders and different types of cancer in mammalian subjects. One ofthese methods involves the modulation of immune responses in a subject.This includes the down regulation of the immune response system usingprocedures or combinations of procedures for producing and applying anew and unexpected immune modulation termed selective immune downregulation (SIDR). Immunological modulation is an artificially inducedvariation in a subject's immune system in response to the introductionof reagents, procedures and processes. These procedures have beendescribed in detail in U.S. patent application Ser. No. 08/808,629,filed on Feb. 28, 1997, U.S. patent application Ser. No. 10/377,628,filed on Mar. 4, 2003, U.S. application Ser. No. 10/377,603, filed onMar. 4, 2003, U.S. patent application Ser. No. 09/447,704, filed on Feb.28, 1997, U.S. application Ser. No. 10/385,440, filed on May 9, 2001,and U.S. application Ser. No. 09/356,294, filed on Jul. 16, 1999. Eachif the foregoing patents are incorporated by reference in their entiretyin the present application and may further be used in conjunction withthe present invention.

Other methods describe the use of educated or treated cells in thetreatment of a variety of diseases. Specifically, the methods aredirected to the manipulation of the NKT cell population in a subjectthat results in the modulation of the Th1/Th2 balance towardanti-inflammatory or pro-inflammatory cytokine producing cells. Adetailed description of these inventions have been disclosed in U.S.patent application entitled “Educated NKT Cells and Their Uses in theTreatment of Immune-Related Disorders” by Yaron Ilan et al., filed onJun. 25, 2003 (application No. not yet assigned), PCT Application No.IL01/01197, filed on Dec. 24, 2001, and U.S. application Ser. No.10/375,906, filed on Feb. 27, 2003. Each of the foregoing patents isincorporated by reference in its entirety in the present application andmay further be used in conjunction with the present invention.

The present invention provides a new method for the treatment ofimmune-related or immune mediated disorders or diseases, infectiousdiseases, metabolic disorders and different types of cancer in mammaliansubjects, and preferably, human subjects. This method involves theadministration of an intermediary metabolite or a T cell receptor ligandto a subject. Other methods disclosed herein use this administrationstep along with other procedures described in prior patent applicationsincorporated by reference herein. These methods are further described indetail below.

An intermediary metabolite or a T cell receptor ligand is used in thepresent invention for the treatment of disease. The intermediarymetabolite or the T cell receptor ligand may comprise a lipid orconjugated biomolecule. The conjugated biomolecule may in turn comprisea glycolipid, lipoprotein, apolipoprotein, or glycoprotein other thanantibodies, cytokines, or hormones. A glycolipid may comprise amonosaccharide ceramide. A monosaccharide ceramide may comprise aglucosylceramide or galactosylceramide.

Glucosylceramide is a naturally occurring glycolipid consisting ofceramide, to which glucose is attached. A ceramide, which is asphingosine and a fatty acid, is the structural unit common to allsphingolipids. Sphingolipids have a variety of cellular functions. Theseinclude membrane structural roles and cell signaling participation.(Sullard et al., 2000 Journal of Mass Spectrometry 35: 347-353.)Glucosylceramide is made by the enzyme glucosylceramide synthase whichattaches the two molecules together. (see FIG. 1 and FIG. 2). An exampleof a glucosylceramide includes glucocerebroside, or a glucocerebrosideanalogue or derivative.

The genetic disease Gaucher's Disease is characterized by anaccumulation of glucosylceramide. In the treatment of this disorder byappropriate enzyme therapy, the excess glucosylceramide is degraded. Twoside effects of this treatment have been noted. In the course of thistreatment, chronic active hepatitis associated with Hepatitis C virusinfection was exacerbated. Additionally, certain patients (withpre-diabetic conditions) experienced the development of diabeticconditions, indicating an onset of Type II Diabetes. These observationsfurther directly confirm that in human subjects, Glucosylceramide levelsregulate the onset of immune-mediated or immune-regulated disorders ordiseases.

SUMMARY OF THE INVENTION

This invention relates to the use of a naturally occurring, mammalianintermediary metabolite or T cell receptor ligand, for the treatment ofimmune mediated or immune related diseases or disorders, infectiousdiseases, metabolic disorders and cancer in mammalian subjects. In apreferred embodiment, such mammalian subjects are human beings.

This invention provides a process for treating a disease in a mammaliansubject comprising administering to the subject an effective amount of amammalian intermediary metabolite.

This invention further provides a process for treating a disease in amammalian subject comprising administering to said subject an effectiveamount of a T cell receptor ligand.

The present invention also provides a process for treating a disease ina mammalian subject comprising administering to said subject aneffective amount of Glucocerebroside.

Another aspect of the present invention provides for the treatment of adisease in a mammalian subject comprising the ex vivo treating oreducating of cells obtained from the mammalian subject. The cells aretreated or educated with an effective amount of the intermediarymetabolite.

The treated or educated cells are then re-administered to the subject.

Another aspect of the present invention provides for the treatment of adisease in a mammalian subject comprising the ex vivo treating oreducating of cells obtained from the mammalian subject. The cells aretreated or educated with an effective amount of the T cell receptorligand.

The treated or educated cells are then re-administered to the subject.

Yet another aspect of the present invention provides for the treatmentof a disease in a mammalian subject comprising the ex vivo treating oreducating of cells obtained from the mammalian subject. The cells aretreated or educated with an effective amount of Glucocerebroside. Thetreated or educated cells are then re-administered to the subject.

The present invention also relates to the treatment of a disease in amammalian subject comprising the re-administration of treated oreducated cells to the subject, and the direct administration to saidsubject of an effective amount of intermediary metabolite.

The present invention provides for the treatment of a disease in amammalian subject comprising the re-administration of treated oreducated cells to the subject, and the direct administration to saidsubject of an effective amount of T cell receptor ligand.

The present invention also relates to the treatment of a disease in amammalian subject comprising the re-administration of treated oreducated cells to the subject, and the direct administration to saidsubject of an effective amount of Glucocerebroside.

Numerous other aspects and embodiments of the present invention aredescribed in further detail below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the chemical structure of Glucocerebroside.

FIG. 2 shows the pathway to Glucosylceramide synthesis.

FIG. 3 shows the effect of Glucocerebroside on liver enzymes.

FIG. 4 shows liver histological sections prepared from mice.

FIG. 5 shows the effect of Glucocerebroside on Serum IFNγ.

FIG. 6 shows the effect of Glucocerebroside on Serum IL2.

FIG. 7 shows the effect of Glucocerebroside on Serum IL12.

FIG. 8 shows the effect of Glucocerebroside on Serum IL-4.

FIG. 9 shows the effect of Glucocerebroside on Serum IL10.

FIG. 10 shows the effect of Glucocerebroside on liver NKT cells.

FIG. 11 shows the effect of Glucocerebroside on spleen NKT cells.

FIG. 12 shows the effect of Glucocerebroside on NKT cell proliferationin vitro.

FIG. 13 shows colonic histological sections prepared from mice.

FIG. 14 shows the effect of Glucocerebroside on Macroscopic ColitisScore.

FIG. 15 shows the effect of Glucocerebroside on Microscopic ColitisScore

FIG. 16 shows the effect of Glucocerebroside on spleen CD4/CD8 ratio.

FIG. 17 shows the effect of Glucocerebroside on liver CD4/CD8 ratio.

FIG. 18 shows the effect of Glucocerebroside on serum cytokine levels.

FIG. 19 shows a Glucose Tolerance Test for Glucocerebroside treatment.

FIG. 20 shows a Glucose Tolerance Test for Glucocerebroside treatment.

FIG. 21 shows the effect of Glucocerebroside on tumor size.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for the treatment of a disease ina mammalian subject by the administration of an effective amount of anintermediary metabolite to the subject. The intermediary metaboliteincludes, but is not limited to a Tcell receptor ligand, a lipid, apolar lipid, a conjugated biomolecule, a glycolipid, a lipoprotein, anapolipoprotein, a glycoprotein, a monosaccharide or polysaccharideceramide, a glucosylceramide, a galactosylceramide, a glucocerebroside,a glucocerebroside analogue or derivative, a sphingosine, a sphingolipidor a ceramide. In a preferred embodiment of the invention, the mammaliansubject is a human being.

The present invention describes a method for treating a disease whereregulatory, immune-regulatory or NKT cells are obtained from the subjectto be treated, or from another subject, and are educated or treated exvivo. The cells are treated or educated by the presence of intermediarymetabolite, antigens or epitopes, and antigen presenting cells, or anycombination thereof. The treated or educated cells are thenre-administered to the subject. The cells may be administered to thesubject by adoptive transfer.

In addition to the method described above involving the ex vivotreatment or education of cells, the present invention also provides fora method where the ex vivo treatment or education is accompanied by themethod of directly administering to the subject to be treated, by avariety of ways, an effective amount of the intermediary metabolite,antigen presenting cells, and antigens or epitopes, or any combinationof the above. The disease may also be treated by only the directadministration of an effective amount of the intermediary metabolite,antigen presenting cells, and antigens or epitopes, or any combinationof the above.

A therapeutic composition for the use in the treatment of the diseasemay comprise an effective amount of the intermediary metabolite, antigenpresenting cells, and antigens or epitopes, or any combination of theabove.

The treatment of a disease in any of the described methods results in achange in the number or function of regulatory, immune-regulatory or NKTcells. This change encompasses a reduction, inhibition, or decrease inthe number or function of the cells. This inhibition may be caused bythe competitive displacement of activating elements from the CD1dmolecule. A change may also include a stimulation or increase in thenumber or function of the cells. This stimulation may be caused byincreased binding of the activating elements from the CD1d molecule.

The treatment of a disease may also result in a change the cytokineresponses. Any cytokine in the immune system may be involved in theseresponses. The change could result in a pro-inflammatory or ananti-inflammatory response. There may also be a pro-inflammatory, and ananti-inflammatory response since certain cytokines may increase andothers may decrease, simultaneously.

Another result of the treatment of a disease is an alteration of theregulatory, immune-regulatory or NKT cell distribution in the subject.This change may also be accompanied by a change in theperipheral/intrahepatic T cell ratio. A further result may also includea change in intrahepatic CD8+ T cell trapping. There may be an increaseor a decrease in the intrahepatic trapping. The result may also includea change in intrasplenic T cell trapping, where said change could be anincrease or decrease.

Also provided in the present invention are two in vitro screening assaysfor an analogue or derivative of an intermediary metabolite which isadministered to the subject to treat a disease. The first assay involvesproviding regulatory, immune-regulatory or NKT cells from the subjectbeing treated or another subject, antigen presenting cells, and ananalogue or derivative of the intermediary metabolite in vitro. If adecrease in the regulatory, immune-regulatory or NKT cell proliferationis identified, then that specific analogue or derivative is a treatmentfor disease.

The second assay involves providing in a first test tube, regulatory,immune-regulatory or NKT cells and BSA; in a second test tube,regulatory, immune-regulatory or NKT cells and the analogue orderivative of an intermediary metabolite; in a third test tube,regulatory, immune-regulatory or NKT cells, antigen presenting cells andBSA; and in a fourth test tube, regulatory, immune-regulatory or NKTcells, antigen presenting cells and the analogue or derivative of theintermediary metabolite. If the least amount of regulatory,immune-regulatory or NKT cell proliferation is found in the fourth testtube, then that specific analogue or derivative is a treatment for thedisease.

The methods for carrying out the invention, and the experimental resultswhich support and further explain the results obtained are as follows:

EXAMPLES I. Glucocerebroside Treatment of Concanavalin-A HepatitisMaterials and Methods Reagents

Concanavalin A was purchased from Worthington biochemical corporation,USA.

Animals

Five groups of male Balb/C mice (n=6/group) were studied.

Serum Transaminase Measurement

Serum ALT and AST plasma activity were measured by an automatedcommercial kit (Kodak SMA).

Hepatic Histology Examination

Histological sections of the livers from all mice were examined todetermine the degree of liver damage. For each mouse a single liversegment was fixed in 10% buffered formaldehyde and embedded in paraffinfor histologic analysis. Sections were stained with hematoxylin/eosinand histologic evaluation was performed.

Measurement of Cytokine Levels

Blood was drawn from mice in all groups and centrifuged at 14,000 rpm.Serum IFNγ, IL2, IL4, IL10 and IL-12 levels were measured by “sandwich”ELISA using Genzyme Diagnostics kits (Genzyme Diagnostics, MA).

Splenic and Hepatic Lymphocyte Isolation

Splenocytes were isolated and red blood cells removed as previouslydescribed [Vicari, A. P., et al., Immunology Today 17(2):71 (1996)].Intrahepatic lymphocytes were isolated from all groups of mice at theend of the study, as previously described, with some modifications[Vicari et al., (1996) ibid.; Bleicher, P. A., et al., Science250:679-682 (1990)]. The inferior vena cava was cut above the diaphragmand the liver was flushed with 5 ml of cold PBS until it became pale.The connective tissue and the gall bladder were removed, and livers wereplace in a 10-ml dish in cold sterile PBS. Livers and spleens werecrushed through a stainless mesh (size 60, Sigma Chemical Co., St. LouisMo.). Cell suspension was placed in a 50 ml tube for 3 minutes andwashed twice in cold PBS (1,250×rpm for 10 minutes), and debris wasremoved. Cells were resuspended in PBS, cell suspension was placedthrough a nylon mesh presoaked in PBS, and unbound cells were collected.Cells were washed twice in 45 ml PBS (1,250×rpm in room temperature).For liver and spleen lymphocyte isolation 20 ml of histopague 1077(Sigma Diagnostics, St. Louis, Mo.) were slowly placed underneath thecells suspended in 7 ml of PBS, in a 50-ml tube. The tube wascentrifuged at 1,640 rpm for 15 minutes at room temperature. Cells atthe interface were collected, diluted in a 50-ml tube, and washed twicewith ice-cold PBS (1,250 rpm for 10 minutes). Approximately 1×10⁶cells/mouse liver were recovered. The viability by trypan blue stainingwas more than 95%. Both splenocytes and liver-associated lymphocyteswere isolated from all animals in all experimental groups.

Flow Cytometry Analysis for NKT Lymphocytes in Peripheral Blood

Immediately following intrahepatic and intrasplenic lymphocyteisolation, triplicates of 2-5×10⁴ cells/500 μl PBS were put into Falcon2052 tubes incubated with 4 ml of 1% BSA for 10 minutes, and centrifugedat 1400 rpm for 5 minutes. Cells were resuspended in 10 μl FCS withanti-NK1.1 and anti-CD3 antibodies (Pharmingen, USA) and mixed every 10minutes for 30 minutes. Cells were washed twice in 1% BSA, and kept in4° C. until reading. For the control group, only 5 μl of 1% BSA wasadded. Analytical cell sorting was performed on 1×10⁴ cells from eachgroup with a fluorescence-activated cell sorter (FACSTAR plus, BectonDickinson). Only live cells were counted, and background fluorescencefrom non-antibody-treated lymphocytes were deducted from the levelsobtained. Gates were set on forward- and side-scatters to exclude deadcells and red blood cells. The data were analyzed with Consort 30two-color contour plot program (Becton Dickinson, Oxnard, Calif.), orthe CELLQuest program.

Example 1 Glucocerebroside Amelioration of Concanavalin-A Hepatitis bythe Inhibition of NKT Regulatory Lymphocytes

To evaluate the immune modulatory effect of Glucocerebroside onConcanavalin-A (Con-A) induced hepatitis, five groups of Balb/C mice,consisting of 6 mice each were studied. Group A and Group B were treatedintraperitoneally with 1 μg Glucocerebroside two hours prior to and twohours following, respectively, the intravenous administration of 500 μgof Con-A. Group C mice received only 500 μg of Con-A, and noGlucocerebroside. Group D mice were treated with 1 μg Glucocerebroside,and no Con-A. Group E mice were naïve controls. This is summarized inTable 1.

TABLE 1 Experimental and Control Groups Group ConA (IV 500 ug)Glucocerebroside (IP 1 ug) A + + (2 hours before ConA) B + + (2 hoursafter ConA) C + − D − + E − −Treatment with Glucocerebroside significantly ameliorated Con-A inducedhepatitis, as shown in FIG. 3 by markedly reduced serum AST and ALTlevels. Group A had an ALT level of 57 IU. Group B and Group C had ALTlevels of 420 IU and 801 IU, respectively. Group A had an AST level of143 IU. Group B and Group C had AST levels of 559 IU and 600 IU,respectively. The administration of Glucocerebroside alone in Group Ddid not show a significant change in AST or ALT levels compared to GroupE, the naïve control.As shown in Table 2, treatment with Glucocerebroside two hours beforeCon-A administration in Group A resulted in normal results in almost allbiopsies. Group B and Group C mice showed ischemia, necrosis andapoptosis. As shown in FIG. 4, liver histological sections prepared fromGroup A and Group B mice revealed markedly attenuated damage compared tosections prepared from Group C livers, in which massive hepatocytedamage and characteristic apoptosis related changes were present.

TABLE 2 Effect of Glycocerebroside on Liver Pathology A Normal in almostall biopsies B Ischemia, necrosis, apoptosis C Ischemia, necrosis,apoptosis D Normal E NormalFIG. 5 shows that Glucocerebroside treatment significantly lowered serumIFNγ levels. Group A had approximately 3,725 pg/ml and Group C had 5,620pg/ml. FIG. 6 shows that serum IL2 levels increased with Glucocerbrosidetreatment: Group A had approximately 602 pg/ml and Group C had 206pg/ml. Serum IL12 levels, as shown in FIG. 7, also increased withGlucocerebroside: Group A had approximately 22,250 pg/ml and Group C had10,100 pg/ml. As shown in Figures and 9, respectively, serum IL4 andIL10 levels decreased with Glucocerebroside treatment. According to FIG.8, Group A had a serum IL4 level of approximately 31 pg/ml and Group Chad 37 pg/ml. According to FIG. 9, Group A had a serum IL10 level ofapproximately 8 pg/ml and Group C had 26 pg/ml.As shown in FIG. 10, the effect of Glucocerebroside on immune mediatedhepatitis was associated with a significant decrease in intrahepatic NKTlymphocytes. Such a decrease did not occur with intrasplenic NKTlymphocytes (see FIG. 11).In FIG. 12, the proliferation of NKT cells containing various componentsin vitro were studied. Group A contained NKT cells and BSA; Group Bcontained NKT cells and Glucocerebroside; Group C contained NKT cells,Dentritic Cells and BSA; and Group D contained NKT cells, DendriticCells and Glucocerebroside. The stimulation index decreased from Group Ato Group D. This depicts that there is an overall decrease in NKT cellproliferation. The presence of Glucocerebroside and Dentritic Cells isnecessary for this NKT cell decrease.The administration of Glucocerebroside resulted in the significantamelioration of Con-A hepatitis. This effect was accompanied by asignificant decrease in the IFNγ response. These results suggest thatthe Glucocerebroside effect may be associated with the inhibition ofintrahepatic NKT cells by the competitive displacement of activatingelements from the CD1d molecule.

II. Glucocerebroside Treatment of Colitis Materials and Methods Animals

Normal inbred 2 to 4 month old Balb/c male mice were obtained fromJackson Laboratories, USA and maintained in the Animal Core of theHadassah-Hebrew University Medical School. Mice were maintained onstandard laboratory chow and kept in 12-hour light/dark cycles.

Induction of Colitis

2,4,6-trinitrobenzene sulfonic acid (TNBS)—colitis was induced by rectalinstillation of TNBS, 1 mg/mouse, dissolved in 100 ml of 50% ethanol asdescribed. [Collins, C., et al., Eur. J. Immunol. 26:3114-3118 (1996)].

Evaluation of the Effect of Glucocerebroside on Experimental Colitis

The effect of Glucocerebroside was evaluated by monitoring the followingparameters for colitis:

Clinical Assessment of Colitis:

Diarrhea was followed daily throughout the study.

Macroscopic Score of Colitis

Colitis assessment was performed 14 days following colitis inductionusing standard parameters [Madsen, K. L., et al., Gastroenterology113:151-159 (1997); Trop, S., et al., Hepatology 27:746-755 (1999)].Four macroscopic parameters were determined, namely: diarrhea, degree ofcolonic ulcerations; intestinal and peritoneal adhesions; and wallthickness. Each parameter was graded on a scale from 0 (completelynormal) to 3 (most severe) by two experienced blinded examiners.

Grading of Histological Lesions

For histological evaluation of inflammation, distal colonic tissue (last10 cm) was removed and fixed in 10% formaldehyde. Five paraffin sectionsfrom each mouse were then stained with hematoxylin-eosin by usingstandard techniques. The degree of inflammation on microscopic crosssections of the colon was graded semiquantitatively from 0 to 4 [Madsenet al., (1997) ibid.; Trop et al., Hepatology 27:746-755 (1999)]. Grade0: normal with no signs of inflammation; Grade 1: very low level ofleukocyte infiltration; Grade 2: low level of leukocyte infiltration;and Grade 3: high level of infiltration with high vascular density, andbowel wall thickening; Grade 4: transmural infiltrates with loss ofgoblet cells, high vascular density, wall thickening, and disruption ofnormal bowel architecture. The grading was performed by two experiencedblinded examiners.

Splenic and Hepatic Lymphocyte Isolation

Splenocytes were isolated and red blood cells removed as previouslydescribed [Vicari, A. P., et al., Immunology Today 17(2):71 (1996)].Intrahepatic lymphocytes were isolated from all groups of mice at theend of the study, as previously described, with some modifications[Vicari et al., (1996) ibid.; Bleicher, P. A., et al., Science250:679-682 (1990)]. The inferior vena cava was cut above the diaphragmand the liver was flushed with 5 ml of cold PBS until it became pale.The connective tissue and the gall bladder were removed, and livers wereplace in a 10-ml dish in cold sterile PBS. Livers and spleens werecrushed through a stainless mesh (size 60, Sigma Chemical Co., St. LouisMo.). Cell suspension was placed in a 50 ml tube for 3 minutes andwashed twice in cold PBS (1,250×rpm for 10 minutes), and debris wasremoved. Cells were resuspended in PBS, cell suspension was placedthrough a nylon mesh presoaked in PBS, and unbound cells were collected.Cells were washed twice in 45 ml PBS (1,250×rpm in room temperature).For liver and spleen lymphocyte isolation 20 ml of histopague 1077(Sigma Diagnostics, St. Louis, Mo.) were slowly placed underneath thecells suspended in 7 ml of PBS, in a 50-ml tube. The tube wascentrifuged at 1,640 rpm for 15 minutes at room temperature. Cells atthe interface were collected, diluted in a 50-ml tube, and washed twicewith ice-cold PBS (1,250 rpm for 10 minutes). Approximately 1×10⁶cells/mouse liver were recovered. The viability by trypan blue stainingwas more than 95%. Both splenocytes and liver-associated lymphocyteswere isolated from all animals in all experimental groups.

FACS of Intrahepatic and Intrasplenic Lymphocytes for NKT, CD4 and CD8Markers

Immediately following lymphocyte isolation, triplicates of 2-5×10⁴cells/500 μl PBS were put into Falcon 2052 tubes incubated with 4 ml of1% BSA for 10 minutes, and centrifuged at 1400 rpm for 5 minutes.Analysis of lymphocyte subpopulations was performed using anti-NK1.1,anti-CD3, anti-CD4 and anti CD-8 antibodies. Cells were washed twice in1% BSA, and kept in 4° C. until reading. For the control group, only 5μl of 1% BSA was added. Analytical cell sorting was performed on 1×10⁴cells from each group with a fluorescence-activated cell sorter (FACSTARplus, Becton Dickinson). Only live cells were counted, and backgroundfluorescence from non-antibody-treated lymphocytes were deducted fromthe levels obtained. Gates were set on forward- and side-scatters toexclude dead cells and red blood cells. The data were analyzed withConsort 30 two-color contour plot program (Becton Dickinson, Oxnard,Calif.), or the CELLQuest program.

Measurement of Cytokine Levels

Blood was drawn from mice in all groups and centrifuged at 14,000 rpm.Serum IFNγ IL2, IL4, IL10 and IL-12 levels were measured by “sandwich”ELISA using Genzyme Diagnostics kits (Genzyme Diagnostics, MA).

Example 1 Glucocerebroside Amelioration of Experimental Colitis

To evaluate the immune modulatory effect of Glucocerebroside in a murinemodel of experimental colitis, four groups of Balb/c mice, consisting of10 mice each were studied. Group A and Group B mice were challenged withrectal TNBS and Group C and Group D were given normal saline. Group Band Group D mice were intraperitoneally administered 1.5 μg ofGlucocerebroside daily, for 9 days. This is summarized in Table 3.

TABLE 3 Experimental and Control Groups Group TNBS Glucocerebroside (IP1.5 ug) A + − B + + C − − D − +

As shown in FIG. 14, treatment with Glucocerebroside showed improvementin the macroscopic colitis score for Diarrhea. Group A had a score ofapproximately 0.22 and Group B had a score of approximately 0.5. Thescore for the degree of colonic ulcerations also improved, since Group Ahad an approximate score of 0.11 and Group B had an approximate score of0.2. There was also a slight improvement in macroscopic score for wallthickness, since both Group A and Group B had approximate scores of 2.44and 2.56, respectively. However, intestinal and peritoneal adhesionsincreased for Group A, versus Group B, where approximate scores were2.56 and 1.4, respectively.

As shown in FIG. 13 and FIG. 15, Group A, which did not receiveGlucocerebroside, had the highest microscopic colitis score ofapproximately 3.6, evidencing a high degree of inflammation. Group B, Cand D had practically normal biopsies (lower microscopic scores).

The administration of Glucocerebroside resulted in marked alleviation ofcolitis, manifested by significant improvement of the macroscopic andmicroscopic colitis scores in Group A mice compared to Group B mice.The effect of Glucocerebroside on Group C and Group D mice showed aSpleen CD4/CD8 ratio of 3.0 and 1.89, respectively. The effect ofGlucocerebroside on Group C and D mice showed a liver CD4/CD8 ratio of8.8 and 3.4, respectively. The ratio of ratios of Group C mice (naïveanimals) versus Group D mice (animals treated with Glucocerebroside)were 0.34 and 0.65, respectively. These results show a decrease in NKTcells in the periphery and the liver, and a decreased CD4/CD8 ratio inthe periphery and the liver. Therefore, the effect of Glucocerebrosidewas more intrahepatic CD8 trapping. These results are shown in FIG. 16and FIG. 17.The effect of Glucocerebroside on Group A and Group B mice showed aSpleen CD4/CD8 ratio of 1.89 and 3.33, respectively. The effect ofGlucocerebroside on Group A and Group B mice showed a liver CD4/CD8ratio of 5.0 and 5.24, respectively. The ratio of ratios of Group A mice(animals with colitis not treated with Glucocerebroside) versus Group Bmice (animals with colitis treated with Glucocerebroside) were 0.34 and0.65, respectively. These results show an increase in NKT cells in theperiphery and no change in NKT cells in the liver. There was anincreased peripheral CD4/CD8 ratio and a mild increase of the liverCD4/CD8 ratio. These results are also shown in FIG. 16 and FIG. 17.Glucocerebroside treatment resulted in more intrahepatic CD8 trapping.FIG. 18 shows the effect of Glucocerebroside on serum cytokine levels.Serum IFNγ levels were increased with Glucocerebroside treatment. GroupA had approximately 8.3 pg/ml and Group B had approximately 27.1 pg/ml.Serum TNFa levels also increased with Glucocerbroside treatment: Group Ahad approximately 75 pg/ml and Group B had approximately 103.6 pg/ml.Serum IL4 levels also increased with Glucocerebroside: Group A hadapproximately 5.7 pg/ml and Group B had approximately 9.1 pg/ml.However, serum IL10 levels decreased with Glucocerebroside treatment.Group A had a serum IL10 level of approximately 42.1 pg/ml and Group Bhad approximately 21.4 pg/ml.Alleviation of colitis by Glucocerebroside treatment was associated witha significant increase in intrahepatic CD8⁺ T cell trapping. Theperipheral/intrahepatic CD4⁺/CD8⁺ ratio increased by 85% in Group A micetreated with Glucocerebroside versus untreated Group B mice. A similareffect was observed when Glucocerebroside was administered to naïveanimals: the peripheral/intrahepatic CD4⁺/CD8⁺ ratio increased by 61% inGroup C Glucocerebroside treated mice versus untreated animals. WhileGlucocerebroside treatment led to a 108% increase of theperipheral/intrahepatic NKT cell ratio in naïve mice, the beneficialeffect of Glucocerebroside on TNBS colitis was associated with arelative decrease of this ratio.Similar results were obtained when the same experiment was conductedwith the 15 μg of Glucocerebroside, administered orally. There was amarked alleviation of colitis manifested by a significant improvement ofthe macroscopic and microscopic colitis scores in Group AGlucocerebroside treated mice compared to the untreated Group B mice, asshown in Table 4.

TABLE 4 Microscopic and Macroscopic Results of the Oral Administrationof Glucocerebroside for the Treatment of Colitis B D Mouse A (TNBS + C(Naïve + No. (TNBS) 15 μgGC) (Naïve) 150 μgGC) Microscopic 1 3 0.5 0.5 12 3.5 1.5 1.5 1 3 0.5 0.5 4 2 0.5 2 5 4 1.5 1.5 0 6 2 1.5 0.5 0 7 2.5 00.5 0.5 8 4 1 0 9 2 0.5 0 10 2 1 Macroscopic 1 0 0.5 0 0.5 2 0.5 0 0 0.53 1 0.5 0 0 4 1.5 0.5 0 0 5 1.5 0.5 0 0 6 3.5 0.5 0 0 7 2.5 0.5 0 0 8 00 0 9 0 0 0 10 0 0 0

III. Glucocerebroside Treatment of Non-Alcoholic SteatohepatitisMaterials and Methods Animals

Ten-week-old male leptin-deficient C57BL/6J mice and lean C57BL/6 micewere purchased from Harlan laboratories and maintained in the AnimalCore of the Hadassah-Hebrew University Medical School. Mice were fedstandard laboratory chow and kept in 12-hour light/dark cycles.

Glucose Tolerance Test

Glucose tolerance was assessed by oral administration of glucose (1 gramper kilogram body weight). Blood drawn from the tail was measured forglucose at 0′, 15′, 30′, 60′, 90′, 120′ and 180′. Glucose levels weremeasured with Elite glucose test strips and a glucometer.

Hepatic MRI Measurement of Fat Content

Hepatic fat content was measured using a double-echo chemical shiftgradient-echo magnetic resonance imaging (MRI) sequence that providesin-phase and opposed-phase images in a single acquisition forassessment/quantification of fat in mouse liver. The T1-weightedopposed-phase MR imaging technique is sensitive for detection ofrelatively small amounts of tissue fat. MRI images were performed with a1.5-T system (Signa LX;GE, Milwaukee, USA). Double-echo MR imaging wasperformed with a repetition time (TR) of 125 msec, double echo times(TEs) of 4 and 6.5 msec, and a flip angle of 80°. Imaging parametersincluded section thickness of 3 mm, 13-cm field of view, 256*160 matrix,and one signal acquired, with use of a knee coil. Transverse (axial) andcoronal images were acquired at the level of the liver with a 3 mmsection thickness and no intersection gap. Quantitative assessment ofsignal intensity (SI) measurements of SI changes between in-phase andopposed-phase images was computed as described in previous reports(Mitchell D G et al., Invest. Radiol 26:1041-1052 (1991); Tomohiro N etal., Radiology 218:642-646 (2001)). The SI index was calculated asfollows: SI index=(SI_(ip)−Si_(op))/SI_(ip), where SI_(ip) is SI onin-phase images and SI_(op) is SI on opposed-phase images. The SI indexreflects the fraction of SI loss on opposed phase images compared withthe SI on in-phase images.

Example 1 Effect of Glucocerebroside on Diabetes

To evaluate the effect of Glucocerebroside on the various metabolic andimmunologic components of the NASH model, four groups of C57b1 mice,consisting of 12 mice each were studied. As shown in Table 5, Group Aand Group B mice were ob/ob mice, whereas Group C and Group D mice werenot. Group A and Group C mice were injected intraperitoneally with 1.5μg in 100 μl PBS every other day for 14 days. Group B and Group D naïveob/ob mice and naïve C57b1 mice, respectively, were left untreated.

TABLE 5 Experimental and Control Groups A OB/OB MICE INJECT WITHGLUCOCEREBROSIDE IP 1.5 μg/mouse in 100 μl PBS every other day B NaiveOB/OB MICE untreated C C57bl INJECT WITH GLUCOCEREBROSIDE IP 1.5μg/mouse in 100 □l PBS every other day D Naive C57bl untreated

On the 14th day, glucose tolerance tests were performed on 6 mice fromeach group. As depicted in FIG. 19, Group A mice, which were treatedwith Glucocerebroside, had a higher glucose tolerance than naïve ob/obmice that were not treated. This suggests that Glucocerebrosideinjection alters the metabolic profile of ob/ob mice, improving theirglucose tolerance results, rendering them less diabetic.

Example 2 Effect of Orally Administered Glucocerebroside on NASH

To evaluate the effect of Glucocerebroside on the various metabolic andimmunologic components of the NASH model, four groups of C57b1 mice,consisting of 12 mice each were studied. As shown in Table 6, Group Aand Group B mice were ob/ob mice, whereas Group C and Group D mice werenot. Group A and Group C mice were injected intraperitoneally with 1.5μg in 100 μl PBS every other day for 14 days. Group B and Group D naïveob/ob mice and naïve C57b1 mice, respectively, were left untreated.

TABLE 6 Experimental and Control Groups A OB/OB MICE FEEDGLUCOCEREBROSIDE 15 μg/mouse in 100 μl PBS every other day B Naive OB/OBMICE untreated C C57bl FEED GLUCOCEREBROSIDE 15 μg/mouse in 100 μl PBSevery other day D Naive C57bl untreated

On the 14th day, glucose tolerance tests were performed on 6 mice fromeach group. As depicted in FIG. 20, Group A mice, which were treatedwith Glucocerebroside, had a higher glucose tolerance than naïve ob/obmice that were not treated. This suggests that immune modulation throughoral immune regulation induction alters the metabolic profile of ob/obmice, improving their glucose tolerance results, rendering them lessdiabetic.

Example 3 The Effect of Glucocerebroside on the Hepatic Fat Content

To determine the effect of Glucocerebroside on the various metabolic andimmunologic components of the NASH model, four groups of C57bl mice,consisting of 12 mice each were studied. As shown in Table 7, Group Aand Group B mice were ob/ob mice, whereas Group C and Group D mice werenot. Group A and Group C mice were injected intraperitoneally with 1.5μg in 100 μl PBS every other day for 14 days. Group B and Group D naïveob/ob mice and naïve C57b1 mice, respectively, were left untreated.

TABLE 7 Experimental and Control Groups A OB/OB MICE INJECT WITHGLUCOCEREBROSIDE IP 1.5 μg/mouse in 100 μl PBS every other day B NaiveOB/OB MICE untreated C C57bl INJECT WITH GLUCOCEREBROSIDE IP 1.5μg/mouse in 100 □l PBS every other day D Naive C57bl untreated

To determine hepatic fat content, mice of all four groups underwent anabdominal MRI on day 14 of the experiment (Table 8). Hepatic fat contentwas determined and was described as the SI index (IP−OP/IP). Liver size,in area, was also determined. The results showed a reduction in liverfat content due to Glucocerebroside treatment. Group A mice treated withGlucocerebroside had an SI index of 0.46, as compared to Group B, whichhad an SI index of 0.54. There was also a reduction in liver sizeresulting form Glucocerebroside treatment. Glucocerebroside treatedGroup A mice had a liver area of 20.14, as compared to Group B, whichhad a liver area of 24.2.

TABLE 8 In Opposite FAT SI INDEX Phase Phase CONTENT (IP − OP/ ImagesImages (IP − QP) IP) Area Thin 518 423 95 0.18 10.5 517 434 83 0.16 11.5476 397 79 0.17 11.5 1040 813 227 0.22 10 892 731 161 0.18 10 Average0.18 10.7

Calculated MRI Hepatic Fat Content of the Six Mice Groups

In Opposite FAT SI INDEX Phase Phase CONTENT (IP − QP/ Images Images (IP− OP) IP) Area Fat 536 351 185 0.35 25 603 293 310 0.51 16 575 251 3240.56 20.5 554 234 320 0.58 23.5 520 202 378 0.61 30.5 560 201 359 0.6428.5 Average 0.54 24.2

In Opposite FAT SI INDEX Phase Phase CONTENT (IP − OP/ Images Images (IP− OP) IP) Area Fat + Tx 514 279 235 0.46 13.5 527 256 271 0.51 20 574305 269 0.47 26 561 344 217 0.39 18.5 462 283 179 0.39 21.5 579 309 2700.47 27.5 1132 502 629 0.56 14 Average 0.464286 20.14286This suggests that Glucocerebroside alters the metabolic profile in away which results in a reduction in the rate of fat accumulation andNASH in the livers of susceptible mammals.

In Opposite FAT SI INDEX Phase Phase CONTENT (IP − OP/ Images Images (IP− OP) IP) Area Thin + Tx 547 479 68 0.12 16.5 443 424 19 0.04 14 472 40963 0.13 8 507 440 67 0.13 16.5 532 438 94 0.18 5.5 534 481 53 0.1 10 987871 117 0.12 15 974 839 135 0.14 13.5 930 870 60 0.06 9 302 787 115 0.138 927 889 40 0.04 15 910 887 23 0.03 10 Average 0.1 11.75

IV. Glucocerebroside Treatment of Melanoma Materials and Methods Animals

Four groups of C57b1 m ice were studied.

Histology Examination

Histological sections of the lungs from mice were examined to determinethe degree of lung damage. For each mouse a single lung segment wasfixed in 10% buffered formaldehyde and embedded in paraffin forhistologic analysis. Sections were stained with hematoxylin/eosin andhistologic evaluation was performed.

Example 1 Effect of Glucocerebroside Treatment on Melanoma

To evaluate the effect of Glucocerebroside on melanoma, four groups ofC57bl mice, consisting of 8 mice each were studied. Group A and Group Bwere subcutaneously administered 1×10⁶ cells of the B16 melanoma cellline and Group C and Group D were intravenously treated with 1×10⁵ cellsof the B16 melanoma cell line to induce melanoma. Group A and Group Cwere treated with 1 μg of Glucocerebroside intraperitoneally, every day,skipping the last two days of every week, starting on the second day ofthe first week. Group B and Group D mice were given saline only. This issummarized in Table 9.

TABLE 9 Experimental and Control Group Group: Melanoma A GC treatment SCB SALINE SC C GC treatment IV D SALINE IVTreatment with Glucocerebroside significantly ameliorated tumor size.Tumors were removed and subsequently measured. The average tumor weightin Group A was 1.63+/−0.82 g, and the average tumor weight in Group Bwas 2.89+/−0.01 g. The differences in tumor size can be seen in FIG. 21.Treatment with Glucocerebroside also showed a decrease in lungmetastasis. Lung cells of Group C and Group D were fixed forhistological analysis. The average number of lung metastasis in Group Cwas 3+/−1 per lung and the mean number of lung metastasis in Group D was8+/−3 per lung.

1. A method for improving glucose tolerance in a mammal, comprising:administering to a mammalian subject in need of increased glucosetolerance a pharmaceutical composition comprising an amount ofglucocerebroside effective to increase glucose tolerance in the mammal,wherein as a result of said administration glucose tolerance in themammal is increased.
 2. The method of claim 1, wherein the mammaliansubject is a human subject.
 3. The method of claim 1, wherein saidadministering step comprises parenteral administration of saidpharmaceutical composition.
 4. The method of claim 3, wherein themammalian subject is a human subject.
 5. The method of claim 1, whereinsaid administering step comprises intravenous, intraperitoneal, orsubcutaneous administration of said pharmaceutical composition.
 6. Themethod of claim 5, wherein the mammalian subject is a human subject. 7.The method of claim 1, wherein said administering step comprises enteraladministration of said pharmaceutical composition.
 8. The method ofclaim 7, wherein the mammalian subject is a human subject.
 9. The methodof claim 1, wherein said administering step comprises oraladministration of said pharmaceutical composition.
 10. The method ofclaim 9, wherein the mammalian subject is a human subject.